Modulating calmodulin binding specificity through computational protein design

Julia M. Shifman, Stephen L. Mayo

Research output: Contribution to journalArticlepeer-review

96 Scopus citations


We report the computational redesign of the protein-binding interface of calmodulin (CaM), a small, ubiquitous Ca2+-binding protein that is known to bind to and regulate a variety of functionally and structurally diverse proteins. The CaM binding interface was optimized to improve binding specificity towards one of its natural targets, smooth muscle myosin light chain kinase (smMLCK). The optimization was performed using optimization of rotamers by iterative techniques (ORBIT), a protein design program that utilizes a physically based force-field and the Dead-End Elimination theorem to compute sequences that are optimal for a given protein scaffold. Starting from the structure of the CaM-smMLCK complex, the program considered 1022 amino acid residue sequences to obtain the lowest-energy CaM sequence. The resulting eight-fold mutant, CaM_8, was constructed and tested for binding to a set of seven CaM target peptides. CaM_8 displayed high binding affinity to the smMLCK peptide (1.3 nM), similar to that of the wild-type protein (1.8 nM). The affinity of CaM_8 to six other target peptides was reduced, as intended, by 1.5-fold to 86-fold. Hence, CaM_8 exhibited increased binding specificity, preferring the smMLCK peptide to the other targets. Studies of this type may increase our understanding of the origins of binding specificity in protein-ligand complexes and may provide valuable information that can be used in the design of novel protein receptors and/or ligands.

Original languageAmerican English
Pages (from-to)417-423
Number of pages7
JournalJournal of Molecular Biology
Issue number3
StatePublished - 2002
Externally publishedYes

Bibliographical note

Funding Information:
This work was supported by the Howard Hughes Medical Institute, the Ralph M. Parsons Foundation, an IBM Shared University Research Grant (to S.L.M.), an NIH postdoctoral fellowship, and the Caltech Initiative in Computational Molecular Biology, awarded by the Burroughs Wellcome Fund (to J.M.S.). We thank K. Beckingham for providing a plasmid containing WT CaM and M. Ary for assistance with the manuscript.


  • Binding affinity
  • Binding specificity
  • Calmodulin
  • Protein design
  • Protein-ligand interactions


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